The development of retroviral vectors capable of specific entry into targeted cell types is key for the clinical use of retroviral vectors for gene delivery and/or gene therapy. The research in this proposal focuses on critical aspects in the generation of efficient retargeted retroviral vectors. We have developed a new system to screen for selective retroviral entry into targeted cells. Through our studies of retroviral entry, a region of the receptor binding domain of feline leukemia virus (FeLV A) was selected for replacement with a random library. Initial proof-of-concept experiments have identified novel independent variants with selective infection into canine osteosarcoma cells and human 293 cells. These isolates fall into two classes. The first utilize receptors within the same family of proteins as the parental FeLV. Recent data indicate that a second class of isolates utilize receptors outside the viral interference group of the parental virus. This indicates that the random retroviral Env library is capable of selecting for variants capable of retargeting entry to novel receptor proteins. Experiments are aimed at testing and defining the potential of this mammalian random Envelope library. Experiments to characterize the new variants with respect to the sequence requirements within the Env insert, the host range of infection, and the receptors utilized are proposed. Improvements to the selection scheme are outlined that will yield a stable Env library of high complexity. The long term objective is to identify retroviral Env isolates capable of efficient entry into limited human tissues. The applications of this system towards gene and cancer therapy are extremely broad. Three classes of transformed cell lines will be screened for targeted entry by the retroviral library. These include renal cell carcinomas, glioblastomas, and small cell lung cancer.